乙型肝炎病毒活性RNase H酶的表达纯化及在药物筛选中的应用

摘要

Objective To express and purify active HBV RNase H in vitro candidate for drug screening.Methods Codon-optimized cDNA sequences for genotype C HBV RNase H was cloned by gene synthesis into pMAL-c5xHis with a N-terminal MBP tag and C-terminal hexahistidine tag.HBV RNase H was expressed in E.coli BL21 cells.RNase H proteins were enriched by nickel-agarose affinity chromatography and identified through SDS-PAGE gel electrophoresis and Western blot.An oligonucleotidedirected RNA cleavage assay was used to measure the RNase H activity,the stability to temperature and the sensitivity to HBV RNase H inhibitor.Results Large amounts of soluble recombinant HBV RNase H was expressed and purified.Purified HBV RNase H could be detected by both Coomassie staining and Western blot analysis.Data demonstrate that the purified HBV RNase H has specific activity and good sensitivity to HBV RNase H inhibitor.Conclusions Recombinant HBV RNase H proteins with specific activity can be expressed in E.coli and enriched by nickel-agarose affinity chromatography,and can be used for biochemical screening of HBV RNase H inhibitors.%目的 获得具有体外活性并且能够用于药物筛选的乙型肝炎病毒(HBV) RNaseH酶.方法 以pMAL-c5xHis为骨架,构建重组基因C型HBV RNase H酶表达质粒,通过大肠埃希菌高效表达出分子量为60KDa的可溶性目的蛋白,通过镍亲和层析法纯化出HBV RNase H酶.Commassie染色和Western Blot方法鉴定目的蛋白;采用寡核苷酸引导的RNA切割法鉴定HBV RNase H酶活性、对温度变化的稳定性及其在HBV RNase H酶抑制剂体外筛选中的应用.结果 Commassie染色、Western Blot分析结合寡核苷酸引导的RNA切割法鉴定显示成功表达和纯化出具有活性的HBVRNase H酶,酶活性对温度变化具有稳定性,并且对HBV RNase H酶抑制剂具有良好的敏感性.结论 通过基因重组和镍亲和层析法可以体外获得具有活性的HBV RNase H酶,并可以用于HBVRNase H酶抑制剂的体外筛选.